Replication of human immunodeficiency virus type 1 (HIV-1) causes AIDS in humans, and antiviral drugs developed by the pharmaceutical industry are currently the most effective way to combat HIV/AIDS. HIV-1 makes three of its own enzymes, reverse transcriptase, protease, and integrase that are essential for its growth. Drugs currently in the clinic target reverse transcriptase and protease, and therefore integrase is the only viral enzyme that is currently untargeted by clinicians. To learn more about the mechanism of integrase action, this application proposes to decipher how the enzyme functions to integrate its cDNA substrate during infection in human cells. During infection, the virus forms a functional preintegration complex comprised of integrase, cDNA, and other viral as well as human cell proteins. Numerous different human proteins have been implicated in the function of HIV-1 preintegration complexes, but it is unclear to what extent these proteins are required during infection. A variety of protein biochemistry and viral genetic experiments will be performed to pinpoint the roles of human cell proteins in HIV-1 integration. Other avenues of research include detailed characterization of preintegration complexes isolated from infected cells. Since each infected cell contains on average just one or two complexes, their detailed characterization has proven difficult. Novel biochemical techniques were proposed to surmount this obstacle, and additional experiments were proposed to construct preintegration complexes from scratch using purified protein and DNA components. The results of these experiments will significantly advance basic knowledge of how HIV-1 accomplishes its integration in human cells, which will contribute to the pharmacological goal of developing drugs that effectively inhibit virus integration during the development of AIDS in humans.